![]() CellProfiler is faster than FociCounter, because it does not require treating each image and cell nuclei individually and it provides more information about the γH2AX foci and the cell nuclei. Open source programs, such as FociCounter and CellProfiler, are used to analyze images after their capture. The programs process the images after capturing them, either manually or automatically. These algorithms permit focus counting, as well as foci size definition. To improve these methodologies, several intelligent computer pattern recognition algorithms have been developed. The second approach is more efficient, but less sensitive, and therefore, it is not suitable for low dose radiation damage assessment. The first method is time-consuming and is subject to the interpretation of the investigator. Quantification of γH2AX foci can be performed either manually, by individually counting the number of foci present in each cell nucleus or automatically, by evaluating the total γH2AX immunofluorescence intensity emission per cell, using high throughput techniques, such as flow cytometry. It thus offers a more complete and rapid assessment of DNA damage. ![]() This system is a reliable tool for γH2AX radio-induced foci counting and provides essential information about the cell cycle stage. The automated scoring was faster than and as sensitive as its manually performed counterpart. Furthermore, restrictive settings of the program classifier reduced the “touching nuclei” problem described in other image analysis software. We could thus distinguish both the number of γH2AX foci per cell and the cell cycle phase. For our purposes, a double staining immunofluorescence was carried out with antibodies to detect γH2AX and pericentrin, an integral component of the centrosome. While scanning the sample, the system classifies the selected nuclei according to the signal patterns previously described by the user. We adapted a FISH (fluorescent in situ hybridization) Spot-counting system, which has a slide loader with automatic scanning and cell capture system throughout the thickness of each cell (z-stack), to meet our assay requirements. The method was set up to measure DNA damage induced in human mammary epithelial cells by irradiation under a mammogram device. Adding this feature to a rapid automated γH2AX foci quantification method would reduce the scoring uncertainty that arises from the variations in the background level of the γH2AX signal throughout the cell cycle. While a few fully automated methods have been described in the literature, none of them have been used to quantify γH2AX foci in combination with a cell cycle phase analysis. In general, both techniques are laborious and prone to artifacts associated with manual scoring. Foci scoring is performed either manually or semi-automatically using hand-operated capturing and image analysis software. Phosphorylation of the H2AX protein is an early step in the double strand break (DSB) repair pathway therefore, phosphorylated histone (γH2AX) foci scoring is widely used as a measure for DSBs.
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